Achieving the national goal of reducing oil imports will require bioenergy production from biomass. Biofuels are secure energy resources because of the domestic and global availability of biomass. Moreover, biofuels are renewable and thereby inexhaustible, expandable to meet the growing demand of national and world economies, and supportable by modern agricultural infrastructure. It has been projected that biological derived energy will make up a substantial portion of future energy use. By 2100, biofuels use could equal all global fossil energy today (8).
The predominant biofuel today is starch-derived ethanol. About 4 billion gallons of ethanol are produced every year, which could displace 2% of fossil fuel consumed (8). However, the expansion of ethanol production from food starch is ultimately limited and has a low 14% energy yield as calculated by energy in ethanol converted from net energy content of the feed-stock. In contrast, cellulose-derived ethanol could have a 37% yield. Furthermore, cellulose and hemicellulose are the major components of plant cell walls and hence the largest components of all biomass. It is estimated that use of waste cellulose can displace 10% of current gasoline usage, and more gasoline usage can be displaced upon development of energy crops.
The current rate-limiting step to produce cellulose-derived ethanol is the degradation of cellulose and hemicellulose polymers to sugars. The rigid, crystalline structure of cellulose and its encasement by hemicellulose and other structural polymer such as lignin prevent the efficient breakdown into sugars. Biomass-degrading microbes and fungi are sources of enzymes to improve wood pre-processing and cellulose breakdown. Many anaerobic bacteria such as Clostridium, Acetivibrio, Bacteroides and Ruminococcus secrete a large extracellular enzyme complex named cellulosome, which is capable of degrading cellulose, hemicellulose and pectin (3, 4, 6, 9, 10). Understanding the molecular mechanisms of these microbial systems is essential for modeling, prediction and engineering of the optimized enzymes and microbes for efficient cellulose conversion.
Current efforts in understanding cellulose degradation by microbes largely focus on several Clostridia strains such as Clostridium thermocellum and Clostridium cellulolyticum. However, the functional studies at molecular level have been frustrated by the lack of efficient genetic tools in these microorganisms. For example, no expression vectors have been reported in C. thermocellum, no mutagenesis systems have been established, and gene transfer technology via electroporation is not straightforward. As to C. cellulolyticum, there is a lack of a targeted mutagenesis method. The only C. cellulolyticum mutants reported so far are spontaneous mutants generated by native transposes existing in the bacterial genome (7). Although it has been expected that some cellulosome components are essential for cellulosome integrity and function in C. cellulolyticum, this hypothesis is largely unproven due to lack of a targeted mutagenesis system. For the same reason, the regulatory systems of cellulosome production and secretion still remain largely unknown. Therefore, there is an urgent need to develop an efficient mutagenesis system for identifying structural and regulatory genes critical for cellulosome function.